In the past decade, laboratories around the world have produced tens of thousands of mutant, transgenic, and wild-type fish lines. Maintaining these valuable genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. One of the major recommendations of the 2007 NCRR workshop 'Achieving High-Throughput Repositories for Biomedical Germplasm Preservation' was for performance of detailed studies to improve sperm cryopreservation for important aquarium fish models. Pursuant to this we propose to establish high-throughput processing of zebrafish sperm for stock centers and laboratories by extending a comprehensive cryopreservation pathway, and by intensifying efficiency and output to enable implementation of germplasm repositories. The Specific Aims are to: 1) broaden the pathway to improve quality and provide multiple access points for samples produced by current approaches, 2) intensify the pathway for automated processing and repository development including use of industrial modeling and decision-making tools, and 3) implement two scalable cryopreservation protocols; the large-scale high throughput method for Resource Center use and, reverse-engineered from it, a small to intermediate scale application for other research laboratories to back up their own lines or submit them to the Resource Center. This will improve quality, reduce variability, and increase efficiency. We propose to improve broodstock conditioning, gamete quality, fertilization and reproduction, and to accommodate the variety of approaches, levels of application, and current and future needs of the research community. This will yield two complementary approaches: one at an industrial scale using automated equipment and pooling of samples to yield the highest level of intensity and efficiency; the other, less intensive but highly reliable, designed or dissemination to other laboratories. The materials generated from this external pathway will feed into the central pathway and will provide an additional form of throughput based on the aggregate production from multiple locations. A high level of standardization will be necessary for both approaches to merge efficiently to deliver high-quality samples into large comprehensive germplasm repositories. Thus, we envision this renewal as a funnel to improve and adapt the wide breadth of technical approaches available for zebrafish cryopreservation, and to focus these activities for repository development that can be eventually coupled with commercially available high-throughput equipment, supplies, and reagents specifically optimized for aquarium fish. This approach is practical and extremely feasible. This renewal proposal builds directly on the success of our established pathway and looks to couple efficiently with future advances.